Herpes simplex virus type 1 (HSV-1) is a neurotropic virus capable of forming latent infections for the lifetime of an individual. Upon stress the viral genome undergoes extensive transcription and replication leading to the production of viral proteins and infectious particles. Contrary to the acute initial infection or subsequent reactivation events, during latency transcription is restricted to a single diploid gene within the long repeat elements of the viral genome. Transcription of this gene generates a family of transcripts known as the latency-associated transcripts (LATs) (see FIG. 1) [Rock, D. L. et al., (1987), J. Virol., 61:3820-3826; Spivack, J. G., and N. W. Fraser, (1987), J. Virol., 61:3841-3847 (Spivack 1987); Stevens, J. G. et al, (1987), Science, 235:1056-1059; and Wechsler, S. L. et al, (1988), J. Virol., 62:4051-4058].
An 8.5 kb LAT (referred to as the minor LAT or MLAT based on its abundance) is postulated on the basis of in situ hybridization of infected tissues and the presence of a LAT promoter element mapping to its 5' end and of a polyadenylation signal near its 3' end. It is found in very low amounts in trigeminal ganglia of infected animals [Mitchell, W. J. et al, (1990), J. Gen. Virol., 71:125-132].
The most abundant LAT species is a 2 kb long transcript (referred to as 2.0 kb LAT), which does not appear to be polyadenylated [Devi-Rao, G. B. et al, (1991), J. Virol., 65:2179-2190; Nicosia, M. et al, (1994), 204:717-728 (Nicosia 1994); Spivack 1987; and Wagner 1988] and also lacks a cap at its 5' end which maps to a splice donor sequence GT [Krause, P. R. et al, (1990) J. Clin. Invest., 86(1):225-241; Krause, P. R. et al, (1991) J. Virol., 65:5619-5623; Spivack, J. G. et al, (1991), J. Virol., 65:6800-6810 (Spivack 1991); and Wagner 1988].
The 2.0 kb LAT is considered to be a unique class of genes, known as the .lambda. class [Spivack 1988]. It has been proposed that the 2.0 kb LAT is a stable intron derived from the larger 8.5 kb MnLAT RNA. Consistent with this, Farrell, M. J. et al, 1991, Proc. Natl. Acad. Sci. USA., 88:790-794 have shown that the 2.0 kb LAT RNA could be spliced out of a .beta.-galactosidase transcript containing the LAT sequences in transient transfections. additionally Wu, T. T. et al, 1996, J. Virol., 70:5962-5967 have recently shown that the majority of the 2.0 kb LAT transcript is in a non-linear structure most likely a lariat. However, the spliced exons of the putative primary transcript MLAT have never been detected.
Removal of a short intron in the 2.0 kb LAT leads to the production of a small variant of 1.5 kb in size. Both transcripts are routinely detectable by Northern hybridization [Rock et al, Spivack 1987, Stevens et al and Wechsler et al, cited above]. These RNAs are partially colinear and are thought to evolve by differential splicing. The 1.5 kb LAT is only observed during latency in neurons, whereas the 2.0 kb LAT is detectable in productive infections in tissue culture and animals with the kinetics of a late gene, as well as during latency [Spivack 1987; Spivack, J. G., and N. W. Fraser, (1988), J. Virol., 62:3281-3287 (Spivack 1988); Wagner, E. K. et al, (1988), J. Virol., 62:1194-1202 (Wagner 1988)].
Two promoters involved in the generation of the 2.0 kb LAT RNA have been identified. They are known as the Latency Active Promoter 1 (LAP 1) [Batchelor, A. H. and P. O'Hare, 1990, J. Virol., 64:3269-3279; Dobson, A. T. et al, 1989, J. Virol., 63:3844-3851; Zwaagstra, J. et al, 1991, Virol., 182:287-297; Zwaagstra et al, 1989, J. Gen. Virol., 70:2163-2169; Zwaagstra et al, 1990, J. Virol., 64:5019-5028] and the Latency Active Promoter 2 (LAP2) [Goins, W. F. et al, 1994. J. Virol., 68:2239-2252]. The LAP1 promoter, mapping to the 5' end of mLAT, is the promoter of this putative transcript. Speculation of a second promoter for the 2.0 kb LAT was prompted by the observation that deletion mutants of the LAT1 promoter still produce 2.0 kb LAT RNA during productive infections in tissue culture and animals, but not during latency [Nicosia, M. et al, 1993, J. Virol., 67:7276-7283]. Subsequently, a second promoter element, LAP2, was mapped at or near the 5' end of the 2.0 kb LAT. This promoter, which lacks a TATA box but has a putative initiator element, may drive transcription of the 2.0 kb LAT RNA during productive infections [Chen, X. et al, 1995, J. Virol., 69:7899-7909; Nicosia 1993]. Studies with virus mutants have revealed that LAP1 operates primarily in latency and LAP2 is mainly active in productive infections [Nicosia, 1993; Chen, 1995 and Dobson 1989]. Since the LAP2 promoter abuts the 5' ends the 2.0 kb LAT, it has been proposed that the 2.0 kb LAT and the 8.5 kb LAT may be separate transcripts generated by transcription from these different promoters [Goins, 1984].
The functions of the LATs have not been clearly determined. Some LAT deletion mutants reactivate with reduced kinetics from latency [Steiner, I. et al, 1989, EMBO J., 8:505-511] and appear to establish latency with reduced efficiency [Sawtell, N. M., and R. L. Thompson, 1992, J. Virol. 66:2157-2169]. However, these phenotypes may not map to the LATs but to other newly identified transcripts that overlap the LAT region [Bohenzky, R. A. et al, 1995, J. Virol., 69:2889-2897; Lagunoff, M. et al, 1996, J. Virol. 70:1810-1817; Lagunoff, M., and B. Roizman, 1994, J. Virol., 68:6021-6028; Lagunoff, M., and B. Roizman, 1995, J. Virol., 69:3615-3623; Singh, J., and E. K. Wagner, 1993, Virol., 196: 220-231; and Yeh, L., and P. A. Schaffer, 1993, J. Virol., 67:7373-82]. During latency, the LATs are nuclear localized; however, in productive infections of tissue culture cells and SCID mice brainstems, the 2.0 kb LAT is present in the cytoplasm. The majority of cytoplasmic 2.0 kb LAT RNA is not associated with polysomes in productively infected tissue culture cells [Nicosia, 1994], suggesting that this transcript is not translated under these conditions.
During natural RNA processing, introns are removed from messenger RNA during a succession of molecular events involving specialized small nuclear ribonucleoproteins (snRNP). Proper recognition of splicing signals such as splice donors (SDs), splice acceptors (SAs) and branchpoint sequences by snRNPs are essential steps in this process. Consensus branch sites are efficiently used whereas non conserved sites are poorly recognized and often lead to suboptimal alternative splicing or exon skipping. Surrounding regions, secondary structures and polypyrimidine tract are also known to influence selection of a branch point. Introns are usually rapidly degraded by a multistep process involving exonucleases, endonucleases, and specialized debranching enzymes. Some stable introns have been described from cellular, viral or bacterial (e.g., thermophilic) origin; however a general mechanism underlying this resistance to degradation is not fully understood.
Different strategies have evolved to confer stability to RNA, particularly mRNA. Known examples include secondary structures serving as protein docking sites, long polyadenylated tails or possibly sequestration in nuclease-free environment. Some resistant introns, including 2.0 kb LAT, are also known to be more resistant to debranching enzymes.
There is a need in the art for methods useful in stabilizing unstable gene transcripts.